Field of the Invention
The present invention relates to an apparatus for confocal observation of a specimen, wherein the specimen particularly comprises biological and/or chemical substances.
Description of Related Art
Known confocal microscopes comprise an illumination device which can be e.g. a laser. With the aid of the illumination device, an illumination radiation is generated which first will be directed onto a mask which can comprise a plurality of openings, e.g. slits or pinhole apertures. Via an objective, the mask will be imaged in the specimen. In this process, an image of the mask is generated in a clearly defined plane of the specimen, i.e. a specimen region. The illumination radiation causes a reaction in the specimen, thus generating emission radiations. Said reaction can be e.g. a fluorescence excitation of markers included in the specimen, or also a simple reflection of the illumination radiations. The emission radiations emitted by the specimen will be returned, again via said objective, to the mask and will pass through the corresponding openings of the mask. With the aid of a beam splitter device, the emission radiations, after passing through the mask device, will be guided out of the optical path toward a detection device such as e.g. a CCD sensor.
When using mask devices with very small openings, the problem exists that only small amounts of illumination radiation will pass through the mask device and reach the specimen. A known approach to cope with this problem is the provision of microlenses upstream of the openings of the mask device, which microlenses will bundle the beams and resp. guide them toward the openings of the mask device. In this arrangement, the beam splitter device for outcoupling the emission radiations is disposed between the mask device and said beam-bundling device, e.g. the lenses.
In order to increase the obtainable information about the specimen under investigation, it is desirable to illuminate the specimen with illumination radiations of different wavelengths since this would make it possible to also generate emission radiations of different wavelengths. This enables e.g. the observation of multiple types of fluorescent markers, which may be selected to be excited at different illumination wavelengths and/or to emit light at different emission wavelengths, and may further be selected to specifically bind to different functional structures of interest in a biological specimen, in a single experiment. A corresponding illumination or examination of the specimen is performed, for example, by using illumination radiations of different wavelengths at the same time, and detecting emission radiation of different wavelengths at the same time as well. Compared to a sequential illumination and detection at the respective multiple wavelengths, this simultaneous examination can significantly accelerate the measurement. It also avoids potential falsification of the measurement results due to photo-bleaching or photo-damage. Such falsification can occur during sequential illumination and detection if the specimen or certain fluorescent markers are bleached or damaged during a first observation at a first illumination wavelength, and hence emit reduced or altered signals during a subsequent second observation using a second illumination wavelength.
A disadvantage of the simultaneous illumination and detection at multiple illumination and emission wavelengths resides in the risk of crosstalk between the emission radiations, i.e. mutual interference of beams of different wavelengths. For example, a first fluorescent marker, designed to be excited at a first illumination wavelength and emit light at a first emission wavelength, may have a sufficiently broad emission spectrum to contribute radiation emission at a second emission wavelength, which is intended for observation of a second fluorescent marker. This would cause the emission of the second fluorescent marker, and hence its concentration, to be systematically overestimated in a parallel observation experiment if the first marker is present in the specimen. A corresponding crosstalk can occur during the excitation of fluorescent markers, if a fluorescent marker designed to be excited at a first illumination wavelength is also excited by illumination radiation of a second wavelength.
It is an object of the invention to provide an apparatus for confocal observation of a specimen which is adapted for simultaneous examination of the specimen by use of illumination radiations of different wavelengths while delivering examination results of good quality.